The present invention generally relates to analytical elements and methods for the selective determination of magnesium. In particular, the present invention relates to carboxy-quinolizines and their use as magnesium indicators.
Magnesium is the most abundant intracellular ion in the body next to potassium, and plays a central role in the biochemistry of all cells. Over three hundred magnesium-dependent enzymes have been identified (See e.g., Elin et al., Am. J. Clin. Pathol. 102: 616-622 [1994]). Magnesium is essential to many physicochemical processes including the activation of ATP in the transfer of energy rich phosphate. This magnesium-dependent ATP phosphorylation is central to cellular energetics and signaling (See, Elin et al., supra; Vescovi et al., Cell 84: 165-174 [1996]; Chelen et al., J. Biol. Chem. 274: 7059-7066 [1999]; Pan et al., J. Biol. Chem. 271: 1322-1328 [1996]; and Agus et al., Ann. Rev. Physiol. 53: 299-307 [1991]). In addition, magnesium plays a vital role in the activation of enzymes involved in lipid, carbohydrate, and protein metabolism, and the preservation of the macromolecular structure of DNA, RNA and ribosomes. Magnesium also has a significant influence on the neuromuscular apparatus. Decreased concentrations of magnesium may result in tetany and convulsions, while increased levels can cause general anesthesia, respiratory failure and cardiac arrest. Because tetany due to reduced magnesium concentrations is clinically indistinguishable from that caused by low calcium levels, it is frequently necessary to perform assays for both serum magnesium and calcium at the same time.
In addition, many epidemiological studies suggest links between plasma free magnesium levels and various diseases. Suggestive correlations have been reported linking magnesium levels to ischaemic heart disease, hypertension, atherosclerosis, osteoporosis, migraines, and many other chronic illnesses (See, Ford et al., Int. J. Epidemiol. 28: 645-651 [1999]; Altura et al., Cell Mol. Biol. Res. 41: 347-359 [1995]; Orimo et al., Ann. NY Acad. Sci. 598: 444-457 [1990]; Sojka et al., Nutrition Rev. 53: 71-74 [1995]; and Ramadan et al., Headache 29: 590-593 [1989]). In general, those correlations have been modest and the results often difficult to reproduce. A major reason for this limitation may be the difficulty of accurately assessing magnesium levels, and particularly the intracellular ionized magnesium concentration.
Because of the important role that magnesium plays in the normal functioning of life processes, it has long been recognized that it is necessary to be able to accurately and reliably measure magnesium levels in the body. Such measurements are particularly useful for the diagnoses and treatment of diseases. Many methods have been used to determine magnesium levels in biological fluids, including precipitation techniques, complexometric titration procedures, dye absorption methods, techniques utilizing cation-selective electrodes, absorption spectrometry, and fluorescent spectrophotometry.
One precipitation method for determining magnesium involves the precipitation of magnesium as magnesium ammonium phosphate. The phosphorus in the precipitate is then quantified by a variety of means, among which are photometric measurement as molybdenum blue or as the molybdivanadate complex. Following determination of the phosphate content, magnesium concentration is calculated mathematically. This method requires the elimination of phosphate contamination of the precipitate and the removal of interfering calcium ion, which results in inaccurate magnesium determinations. Thus, this method is cumbersome, time-consuming, and often lacks accuracy. Another precipitation method involves the precipitation of magnesium with 8-hydroxyquinoline. The precipitate can then be quantitated by titrimetry, colorimetry, flame photometry or fluorometry. As with phosphate precipitation techniques, calcium interference must be eliminated, and these procedures suffer the same drawbacks of being tedious, time-consuming, and lacking in accuracy. In addition, this precipitation technique requires special instrumentation that may not be easily available to smaller laboratories.
Various titrimetric techniques are used for the determination of magnesium based on titrations with the complexing agent EDTA (ethylenediamine-tetraacetic acid) using a variety of indicators such as Eriochrome Black T (3-hydroxy-4-[(1-hydroxy-2-naphthalenyl)azo]-7-nitro-1-naphthalene-sulfonic acid monosodium salt). Since calcium is also chelated by EDTA, calcium concentration must be established, either by a second titration or other means, and the magnesium level then calculated as the difference. However, it is often difficult to produce measurements with much precision or accuracy due to the largely subjective measurements of the color change at the titration endpoint, which may vary according to the rate of titration, or may be gradual in the presence of protein or moderately high levels of phosphate (e.g., as in urine and serum samples derived from patients suffering from renal failure).
Another method for determining magnesium involves dye absorption methods utilizing Titan Yellow. However, the colloidal or unevenly dispersed nature of the magnesium hydroxide-dye cake generally affects the accuracy of this method, resulting in erratic measurements which do not agree with those obtained by atomic absorption. In addition, this method suffers from other drawbacks, including the need for preparing a protein-free filtrate, limited sensitivity, color instability, and significant interference from calcium gluconate which is frequently administered in clinical situations where magnesium levels are being monitored.
Direct calorimetric dye-complexing methods using the indicators Magon, methylthymol blue, and Calmagite have recently become increasingly popular. The use of Magon was first reported by Mann et al., (Mann et al., Anal. Chem. 28: 202-205 [1956]) and by Bohoun (Bohoun, Coin. Chim. Acta 7: 811-817 [1962]). Currently used modifications of these original methods are relatively fast and simple to perform. However, these methods generally suffer from significant interference, and have been shown to produce erroneous results in the presence of citrate, a common constituent of intravenous fluids and blood used for transfusions. In addition, the reagents used in the determination of magnesium by reaction with Calmagite are somewhat unstable.
Atomic absorption is perhaps the most accurate means of determining magnesium levels. When light from a lamp containing a magnesium electrode passes through a flame which contains vapors of a fluid whose magnesium content is to be measured, the amount of light that is absorbed by the flame is directly proportional to the magnesium concentration. The intensity of the emerging light beam passes via a monochromator to a photomultiplier detector. Although this method is generally considered to be the reference method for magnesium determinations, expensive instrumentation required for atomic absorption spectroscopy limits its routine use in the clinical laboratory.
Magnesium ion selective electrodes have recently been used as magnesium indicators (Elin et al., Scand. J. Clin. Lab. Invest. Suppl. 224: 203-210 [1996]; Cecco et al., Am. J. Clin. Pathol. 108: 564-569 [1997[; Huijgen et al., Clin. Chem. Lab. Med. 37: 465-470 [1999]; Hristova et al., Clin. Chem. 43: 394-399 [1997]; and Rehak et al., Clin. Chem. 43: 1395-15600 [1997]). However, ionized magnesium concentrations determined using magnesium selective electrodes have been reported to vary with the concentration of calcium, and with the electrode manufacturer. In studies of serum derived from chronic alcoholics, results for ionized magnesium were found to be instrument dependent, so that the usefulness of the measurement could not be evaluated. Thus, it appears that magnesium selective electrodes for measuring ionized magnesium in serum and plasma give irreproducible results (Hristova et al., supra; Rehak et al., supra; and Csako et al., Eur. J. Clin. Chem. Clin. Biochem. 35: 701-709 [1997]). In addition, thiocyanate present in serum derived from smokers has been reported to interfere with free magnesium determination using ion selective electrodes. In patients with severe hypomagnesemia, values for ionized magnesium determined using ion selective electrodes were found to exceed values for total magnesium in some cases.
Fluorescence spectroscopy is a useful tool in monitoring intracellular levels of magnesium. This methodology is considered non-invasive since only a minute amount of the fluorescent metal chelator is loaded in the cell. The response time of fluorescence lies in the millisecond domain. Thus, ion fluxes induced by an extra-cellular stimulus can be monitored immediately. A commonly used fluorescent indicator is 8-hydroxyquinoline. However, the accuracy of observed results using 8-hydroxyquinoline is easily affected by interference from numerous drugs or medications which also fluoresce, and by interference due to nonspecific random quenching of fluorescence. In addition, 8-hydroxy-quinoline is not readily loaded into cells, due to the lack of a carboxyl group.
As indicated above, currently used methods for determining magnesium levels in biological specimens are generally time consuming, inaccurate, insufficiently selective, and/or rely upon expensive instrumentation which is not likely to be available except in the largest and most highly sophisticated clinical laboratories. Furthermore, many difficulties have hindered the development of accurate and precise methods for the determination of magnesium, such as the nonspecific nature of its precipitation reactions, the susceptibility to interference from other ions, and the relatively low intensity of its spectral lines. Thus, there remains a need for methods of determining magnesium which are accurate, simple and inexpensive.
The present invention generally relates to analytical elements and methods for the selective determination of magnesium. In particular embodiments, the present invention provides methods for detecting magnesium, particularly methods for detecting Mg2+ utilizing carboxy-quinolizines having the structure as shown in FIG. 1, Panel A.
The present invention provides methods for detecting magnesium, comprising the steps of: a) providing: i) a sample suspected of containing magnesium, and ii) a composition comprising a carboxy-quinolizine compound; b) contacting the sample with the composition to provide a complex; and c) detecting a fluorescence in the complex, wherein fluorescence indicates the presence of magnesium in the sample.
In one embodiment, the methods of the present invention provide a carboxy-quinolizine compound substituted with a functional group at the C-8 position. In particular embodiments, the functional group at the C-8 position is selected from the group consisting of chloride, xe2x80x94NH2, xe2x80x94O-aryl-Nxe2x80x94(CH2CO2Me)2, xe2x80x94CH(CO2Me)2, xe2x80x94C(CO2Me)2(CO2Et), p-methoxyphenyl, naphthyl, and benzo-[b]furyl.
In yet another embodiment, the carboxy-quinolizine compound is substituted with a functional group at the C-1 position. In particular embodiments, the functional group at the C-1 position is selected from the group consisting of bromide, xe2x80x94N(CH2CO2Me)2, xe2x80x94CH2xe2x80x94CH(CO2Et)2, and xe2x80x94CHxe2x95x90Cxe2x80x94CO2Et)2.
In yet another embodiment, the methods of the present invention provide a carboxy-quinolizine compound substituted with a functional group at the C-1 and C-8 positions. In particular embodiments, the functional group at the C-1 position is bromide. In other embodiment, the functional group at the C-8 position is selected from the group consisting of xe2x80x94C(CO2Me2)(CO2Et), xe2x80x94CH2CO2H and xe2x80x94CH(CH2CO2H)(CO2H). In still other embodiments, the functional group at the C-1 position is bromide, and the functional group at the C-8 position is xe2x80x94C(CO2Me2)(CO2Et).
In another embodiment, the methods of the present invention provide a carboxy-quinolizine compound comprising at least two carboxyl groups. In other embodiments, the methods of the present invention provide a carboxy-quinolizine compound comprising a triacid.
In still other embodiments, the detecting step in the methods of the present invention further comprises visibly detecting the fluorescence in the complex.
In one embodiment, the methods of the present invention provide a carboxy-quinolizine compound having a Mg2+ dissociation constant of about 1 mM. In another embodiment, the carboxy-quinolizine compound binds selectively to magnesium. In yet another embodiment, the carboxy-quinolizine compound binds selectively to magnesium ion. In yet another embodiment, the carboxy-quinolizine compound fluoresces at a wavelength greater than 500 nm.
In other embodiments, the methods of the present invention provide a sample from a subject suspected of suffering from cardiovascular disease. In another embodiment, the sample is from a subject suspected of suffering from hypertension.
Furthermore, the methods of the present invention provide a carboxy-quinolizine compound selected from the group consisting of 4-oxo-8-chloro-4H-quinolizine-3-carboxylic acid, 4-oxo-8-chloro-4H-quinolizine-3-carboxylic acid, [6,7]-benzo-4-oxo-4H-quinolizine-3-carboxylic acid, 1-bromo-4-oxo-4H-quinolizine-3-carboxylic acid, 1-[N,N-di(carboxymethyl)]-4-oxo-4H-quinolizine-3-carboxylic acid, 1-(2,2-dicarboxyvinyl)-4-oxo-4H-quinolizine-3-carboxylic acid, 1-(2,2-dicarboxyethyl)-4-oxo-4H-quinolizine-3-carboxylic acid, 8-amino-4-oxo-4H-quinolizine-3-carboxylic acid, 8-[3-N,N-di(carboxymethyl)phenoxy]-4-oxo-4H-quinolizine-3-carboxylic acid, 8-carboxymethyl-4-oxo-4H-quinolizine-3-carboxylic acid, 8-(1,2-dicarboxyethyl)-4-oxo-4H-quinolizine-3-carboxylic acid, 1-bromo-8-(1,2-dicarboxyethyl)-4-oxo-4H-quinolizine-3-carboxylic acid, 8-(4-methoxyphenyl)-4-oxo-4H-quinolizine-3-carboxylic acid, 8-(naphth-1-yl)-4-oxo-4H-quinolizine-3-carboxylic acid, and 8-(benzo[b]furyl)-4-oxo-4H-quinolizine-3-carboxylic acid.
The present invention further provides compositions suitable for selective fluorescence detection of magnesium, comprising at least one substituted carboxy-quinolizine. In preferred embodiments, the carboxy-quinolizine is a substituted 4-oxo-4H-quinolizine-3-carboxylic acid. In some preferred embodiments, the carboxy-quinolizine is substituted with a functional group at the C-1 position. In alternative preferred embodiments, the functional group at said C-1 position is selected from the group consisting of bromide, xe2x80x94N(CH2CO2Me)2, xe2x80x94CH2CH(CO2Et)2, and xe2x80x94CHxe2x95x90Cxe2x80x94(CO2Et)2. In still further preferred embodiments, the carboxy-quinolizine is substituted with a functional group at the C-1 and C-8 positions. In yet other preferred embodiments, the functional group at said C-1 position is bromide. In additional preferred embodiments, the functional group at said C-8 position is selected from the group consisting of xe2x80x94C(CO2Me2)(CO2Et), xe2x80x94CH2CO2H and xe2x80x94CH(CH2CO2H)(CO2H). In some particularly preferred embodiments, the functional group at said C-1 position is bromide, and said functional group at the C-8 position is xe2x80x94C(CO2Me2)(CO2Et). In additional preferred embodiments, the carboxy-quinolizine comprises at least two carboxyl groups, while in other preferred embodiments, the carboxy-quinolizine comprises a triacid. In still other particularly preferred embodiments, the carboxy-quinolizine has a dissociation constant of about 1 mM.